Abstract The goal of the proposed experiments is to test the hypothesis that ? (9)-tetrahydrocannabinol (THC), the psychoactive compound in cannabis, impairs human cytotrophoblast (CTB) functions and that the negative consequences on trophoblast function are evident beginning at implantation. Nearly half the States and the District of Columbia have legalized marijuana for recreational or medical use. As a result, over 20 million people in the US report using this drug and the number is rapidly rising. In addition, synthetic THC derivatives with enhanced receptor binding profiles are widely available and gaining in popularity. Substantial numbers of pregnant women also use marijuana?2-30% of mothers depending on the subpopulation. Why are these numbers so high? Many women believe that marijuana, a natural product, is inherently safer than other drugs. Given the magnitude of marijuana use during pregnancy, relatively little is known about the impact on pregnancy. The strongest data are from studies of the endogenous signaling system in animal models. In the mouse, endocannabinoid levels, primarily via interactions with cannabinoid receptor 1 (CB1), govern outcomes, leading to the concept that anandamide (CB1 ligand) ?tone? is an important component of the dialogue between the trophoblast and the uterus. In humans, dysregulated levels of endocannabinoid ligands and their receptors in plasma, trophoblast or decidua have been reported in the setting of spontaneous pregnancy loss and recurrent miscarriage. In line with these data, THC administration to mice impairs many aspects of pregnancy. In humans, the data suggest that marijuana use leads to higher rates of sillbirth, low birth weight, neonatal intensive care admissions and (variably) preterm birth. Recent data from our group might help explain this relationship. Global transcriptional profiling, confirmed by protein level data, showed that invasive cytotrophoblasts (CTBs) expressed CB1. As to function, we made the unexpected discovery that a CB1 agonist significantly increased CTB invasion, an unusual effect. In this context, we propose two Specific Aims. The goal of the first is to establish biologically effective doses of THC in early gestation CTBs. The goal of the second is to identify the molecular targets of CB1 ligands, including THC, in these cells. The proposed work will yield several important results. To date, only 3 published studies have investigated THC effects on the human placenta. Term samples were dosed in vitro and targeted analyses evaluated a limited number of endpoints. To fill this knowledge gap, we propose global transcriptional profiling of THC effects on early gestation CTBs, which will reveal effects on implantation and the formative early stages of pregnancy in which CTBs attach to, invade and remodel the uterus. Finally, this basic science research project has translational potential in terms of yielding information that enables evidence-based decisions regarding the use of marijuana (and synthetic THCs) during pregnancy. Additionally, CB1 agonists that increase CTB invasion could be useful for treating pregnancy disorders, such as preeclampsia, which are associated with shallow placentation.